EGF receptor (EGFR) ligands are produced as type 1 transmembrane proteins that are cleaved from the cell surface to produce soluble ligand. EGFR activation can be achieved by soluble ligand (autocrine or paracrine) or by membrane-tethered ligand (juxtacrine). The major focus of my sole R01 grant (Role of EGFR Ligands in Neoplasia) is the trafficking of these ligands in the context of polarized epithelial cells. In this competing revision, we are requesting support to pursue a novel form of cell-cell signaling mediated by EGFR ligands packaged and secreted as exosomes. These EGFR ligand-containing exosomes, are robust signaling platforms and we have found that they present a highly active form of amphiregulin (AR). Exosomes from MDA-MB-231 and HCA-7 differ in their compostion of EGFR ligands. Exosomes from both lines displayed TGF? and heparin-binding EGF-like growth factor (HB-EGF) whereas only HCA-7 exosomes displayed AR. To explore the function of individual ligands, we isolated exosomes from MDCK cells, which do not express endogenous EGFR ligands, and MDCK cells stably expressing TGF?, HB-EGF or AR. In vitro invasion assays using LM2-4175 recipient cells demonstrated that exosomes derived from AR-expressing cells were 4-fold more active than those from TGF?- and HB-EGF-expressing cells, 13-fold more active than MDCK-AR lysates and 15-30 fold more active than similar concentrations of recombinant human TGF?, HB-EGF and AR. Pretreatment of exosomes displaying AR with AR neutralizing antibody reduced invasion by 50%. These preliminary studies demonstrate for the first time that exosomes contain EGFR ligands and that, in particular, AR-containing exosomes increase invasiveness of recipient cells. We propose that exosomally displayed EGFR ligands can provide both local and distant signals that are distinct from signals generated by soluble EGFR ligands. Based upon these novel findings, we propose the following two new specific aims to provide a detailed characterization of EGFR ligand-containing exosomes with a special emphasis on AR-containing exosomes: Aim 1. To determine whether uptake of AR-containing exosomes by recipient cells is EGFR dependent using isogenic wild-type and EGFR-deficient recipient colonic cells; Aim 2. To characterize exosome production by normal and Ras-transformed colon cancer cells. PUBLIC HEALTH RELEVANCE: Regulated signaling through the EGFR is important in normal physiology, and its dysregulation is a common feature of many cancers. We have identified a novel mode of EGFR ligand signaling, that is, via exosomes;and, in particular, exosomal AR enhances invasiveness of recipient breast cancer cells. These studies provide a new paradigm for cell-cell signaling and have important implications for the pathogenesis and treatment of cancer.